Compositions and methods for labeling  of nucleic acid molecules

ABSTRACT

The present invention is generally related to compositions, kits and methods for labeling nucleic acid molecules using reverse transcriptases, preferably multi-subunit reverse transcriptases such as ASLV reverse transcriptases. Specifically, the invention relates to methods, kits and compositions for fluorescently labeling nucleic acid molecules during nucleic acid synthesis. The labeled nucleic acid molecules produced in accordance with the invention are particularly suited as labeled probes for nucleic acid detection and diagnostics.

FIELD OF THE INVENTION

The present invention is in the fields of molecular and cellularbiology. The invention generally relates to the use of reversetranscriptase (RT) enzymes and particularly to methods for the reversetranscription of nucleic acid molecules, especially messenger RNAmolecules, to synthesize labeled (e.g. fluorescently labeled) nucleicacid molecules. The invention also relates to nucleic acid moleculesproduced by these methods and to the use of such labeled nucleic acidmolecules as detection probes. The invention also concerns kits andcompositions for making such labeled nucleic acid molecules.

BACKGROUND OF THE INVENTION cDNA and cDNA Libraries

In examining the structure and physiology of an organism, tissue orcell, it is often desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is only manifested uponproduction of the protein which the gene encodes. In order to produce aprotein, a complementary copy of one strand of the DNA double helix (the“coding” strand) is produced by polymerase enzymes, resulting in aspecific sequence of ribonucleic acid (RNA). This particular type ofRNA, since it contains the genetic message from the DNA for productionof a protein, is called messenger RNA (mRNA).

Within a given cell, tissue or organism, there exist myriad mRNAspecies, each encoding a separate and specific protein. This factprovides a powerful tool to investigators interested in studying geneticexpression in a tissue or cell—mRNA molecules may be isolated andfurther manipulated by various molecular biological techniques, therebyallowing the elucidation of the full functional genetic content of acell, tissue or organism.

One common approach to the study of gene expression is the production ofcomplementary DNA (cDNA) clones. In this technique, the mRNA moleculesfrom an organism are isolated from an extract of the cells or tissues ofthe organism. This isolation often employs solid chromatographymatrices, such as cellulose or agarose, to which oligomers of thymidine(T) have been complexed. Since the 3′ termini on most eukaryotic mRNAmolecules contain a string of adenosine (A) bases, and since A binds toT, the mRNA molecules can be rapidly purified from other molecules andsubstances in the tissue or cell extract. From these purified mRNAmolecules, cDNA copies may be made using the enzyme reversetranscriptase (RT), which results in the production of single-strandedcDNA molecules. The single-stranded cDNAs may then be converted into acomplete double-stranded DNA copy (i.e., a double-stranded cDNA) of theoriginal mRNA (and thus of the original double-stranded DNA sequence,encoding this mRNA, contained in the genome of the organism) by theaction of a DNA polymerase. The protein-specific double-stranded cDNAscan then be inserted into a plasmid or viral vector, which is thenintroduced into a host bacterial, yeast, animal or plant cell. The hostcells are then grown in culture media, resulting in a population of hostcells containing (or in many cases, expressing) the gene of interest.

This entire process, from isolation of mRNA to insertion of the cDNAinto a plasmid or vector to growth of host cell populations containingthe isolated gene, is termed “cDNA cloning.” If cDNAs are prepared froma number of different mRNAs, the resulting set of cDNAs is called a“cDNA library,” an appropriate term since the set of cDNAs represents a“population” of genes comprising the functional genetic informationpresent in the source cell, tissue or organism. Genotypic analysis ofthese cDNA libraries can yield much information on the structure andfunction of the organisms from which they were derived.

Retroviral Reverse Transcriptase Enzymes

Three prototypical forms of retroviral RT have been studied thoroughly.Moloney Murine Leukemia Virus (M-MLV) RT contains a single subunit of 78kDa with RNA-dependent DNA polymerase and RNase H activity. This enzymehas been cloned and expressed in a fully active form in E. coli(reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press, p. 135 (1993)). HumanImmunodeficiency Virus (HIV) RT is a heterodimer of p66 and p51 subunitsin which the smaller subunit is derived from the larger by proteolyticcleavage. The p66 subunit has both a RNA-dependent DNA polymerase and anRNase H domain, while the p51 subunit has only a DNA polymerase domain.Active HIV p66/p51 RT has been cloned and expressed successfully in anumber of expression hosts, including E. coli (reviewed in Le Grice, S.F. J., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory press, p. 163 (1993)). Within the HIV p66/p51heterodimer, the 51-kD subunit is catalytically inactive, and the 66-kDsubunit has both DNA polymerase and RNase H activity (Le Grice, S. F.J., et al., EMBO Journal 10:3905 (1991); Hostomsky, Z., et al., J.Virol. 66:3179 (1992)). Avian Sarcoma-Leukosis Virus (ASLV) RT, whichincludes but is not limited to Rous Sarcoma Virus (RSV) RT, AvianMyeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) HelperVirus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT,Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, AvianSarcoma Virus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 HelperVirus YAV RT, Rous Associated Virus (RAV) RT, and MyeloblastosisAssociated Virus (MAV) RT, is also a heterodimer of two subunits, α0(approximately 62 kDa) and β (approximately 94 kDa), in which α isderived from β by proteolytic cleavage (reviewed in Prasad, V. R.,Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1993), p. 135). ASLV RT can exist in two additionalcatalytically active structural forms, ββ and α (Hizi, A. and Joklik, W.K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysis suggests αβand ββ are dimers and that the a form exists in an equilibrium betweenmonomeric and dimeric forms (Grandgenett, D. P., et al., Proc. _(—) Nat.Acad. Sci. USA 70: 230 (1973); Hizi, A. and Joklik, W. K., J. Biol.Chem. 252: 2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat.Acad. Sci. USA 85: 3372 (1988)). The ASLV αβ and ββ RTs are the onlyknown examples of retroviral RT that include three different activitiesin the same protein complex: DNA polymerase, RNase H, and DNAendonuclease (integrase) activities (reviewed in Skalka, A. M., ReverseTranscriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress (1993), p. 193). The α form lacks the integrase domain andactivity.

Various forms of the individual subunits of ASLV RT have been cloned andexpressed. These include a 98-kDa precursor polypeptide that is normallyprocessed proteolytically to β α, and a 4-kDa polypeptide removed fromthe β carboxy end (Alexander, F., et_al., J. Virol. 61: 534 (1987) andAnderson, D. et al., Focus 17:53 (1995)), and the mature β subunit(Weis, J. H. and Salstrom, J. S., U.S. Pat. No. 4,663,290 (1987); andSoltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85:3372(1988)). Heterodimeric RSV αβ RT has also been purified from E. colicells expressing a cloned RSV β gene (Chemov, A. P., et al., Biomed.Sci. 2:49 (1991)). See also published PCT application WO 98/47912.

Labeling Nucleic Acid Molecules

As noted above, the conversion of mRNA to cDNA by RT-mediated reversetranscription is an essential step in the study of proteins expressedfrom cloned genes. Reverse transcription of nucleic acid molecules,particularly mRNA, to make labeled nucleic acid molecules (e.g., labeledcDNA) is also important in the generation of labeled probes for use indetection and diagnostics. Typically, fluorescent labels are used in thegeneration of such probes. To date, SuperScript™ II (an RNase H minusderivative of MMLV RT available from Life Technologies, Inc.) has beenused in the generation of fluorescently labeled probes from mRNAtemplates (DeRisi et al., Science 278:680-686 (1997)). However, theincorporation rate of fluorescent nucleotides during synthesis isrelatively low (less than 2%), perhaps due to the inability of MMLV RTto effectively use fluorescently labeled nucleotides as substratesduring nucleic acid synthesis. Accordingly, there exists a need for moreefficient incorporation of labeled nucleotides, particularlyfluorescently labeled nucleotides, during reverse transcription of anucleic acid template. Efficient incorporation of such nucleotides willallow for improved synthesis of labeled probes which may be used in theresearch market as well as in the field of diagnostics.

SUMMARY OF THE INVENTION

The present invention provides reverse transcriptase enzymes,compositions and kits comprising such enzymes, and methods useful inovercoming the above-described nucleic acid labeling limitations. Ingeneral, the invention relates to the use of multi-subunit RTs(particularly heterodimers and more specifically two subunit enzymes(e.g., dimers) such as HIV RT and ASLV RTs) to label synthesized nucleicacid molecules. Preferably, such labeling involves the use of labelednucleotides, particularly fluorescently labeled nucleotides and one ormore nucleic acid templates (preferably RNA and most preferably mRNA).In accordance with the invention, one or more labeled nucleic acidmolecules are synthesized which are complementary to all or a portion ofthe one or more templates. The labeled nucleic acid molecules preferablyhave one or more labeled nucleotides incorporated into the synthesizedmolecule and in a preferred aspect, the labels are one or morefluorescent labels (which may be the same or different).

The invention also relates to compositions for use in the invention andsuch compositions may comprise one or more multi-subunit RTs(particularly HIV and ASLV RTs). Such compositions may further compriseone or more nucleotides, a suitable buffer, and/or one or more DNApolymerases. The compositions of the invention may also comprise one ormore primers. The reverse transcriptases in these compositionspreferably have RNase H activity or are reduced or substantially reducedin RNase H activity, and most preferably are enzymes selected from thegroup consisting of Rous Sarcoma Virus (RSV) reverse transcriptase,Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous AssociatedVirus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV)reverse transcriptase and Human Immunodeficiency Virus (HIV) reversetranscriptase or other ASLV reverse transcriptases. Two subunit RTs arepreferred in the use of the invention and such enzymes may containvarious forms and combinations of such subunits such as αβ, αα, ββ, etc.and mutants, variants or derivatives thereof. In preferred compositions,the reverse transcriptases are present at working concentrations.

The invention is also directed to methods for making one or more labelednucleic acid molecules, comprising mixing one or more nucleic acidtemplates (preferably one or more RNA templates and most preferably oneor more messenger RNA templates) with one or more polypeptides orenzymes having reverse transcriptase activity (preferably one or moremulti-subunit RTs) and incubating the mixture under conditionssufficient to synthesize one or more first nucleic acid moleculescomplementary to all or a portion of the one or more nucleic acidtemplates, wherein said at least one of said synthesized molecules arelabeled and/or comprise one or more labeled nucleotides. In a preferredembodiment, the one or more first nucleic acid molecules aresingle-stranded cDNA molecules. Nucleic acid templates suitable forreverse transcription according to this aspect of the invention includeany nucleic acid molecule or population of nucleic acid molecules(preferably RNA and most preferably mRNA), particularly those derivedfrom a cell or tissue. In a preferred aspect, a population of mRNAmolecules (a number of different mRNA molecules, typically obtained fromcells or tissue) are used to make a labeled cDNA library, in accordancewith the invention. Preferred cellular sources of nucleic acid templatesinclude bacterial cells, fungal cells, plant cells and animal cells.

The invention also concerns methods for making one or moredouble-stranded nucleic acid molecules. Such methods comprise (a) mixingone or more nucleic acid templates (preferably RNA or mRNA, and morepreferably a population of mRNA templates) with one or more polypeptidesof the invention having reverse transcriptase activity (preferably oneor more multi-subunit RTs); (b) incubating the mixture under conditionssufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of the one or more templates; and (c)incubating the one or more first nucleic acid molecules under conditionssufficient to make one or more second nucleic acid moleculescomplementary to all or a portion of the one or more first nucleic acidmolecules, thereby forming one or more double-stranded nucleic acidmolecules comprising the first and second nucleic acid molecules. Inaccordance with the invention, the first and/or second nucleic acidmolecules are labeled (e.g., may comprise one or more of the same ordifferent labeled nucleotides). Thus, labeled nucleotides may be used atone or both synthesis steps. Such methods may include the use of one ormore DNA polymerases as part of the process of making the one or moredouble-stranded nucleic acid molecules. The invention also concernscompositions useful for making such double-stranded nucleic acidmolecules. Such compositions comprise one or more reverse transcriptasesof the invention and optionally one or more DNA polymerases, a suitablebuffer and/or one or more nucleotides (preferably including labelednucleotides).

The invention is also directed to labeled nucleic acid molecules(particularly single- or double-stranded cDNA molecules) producedaccording to the above-described methods and to kits comprising thesenucleic acid molecules. Such molecules or kits may be used to detectnucleic acid molecules (for example by hybridization) or for diagnosticpurposes.

The invention is also directed to kits for use in the methods of theinvention. Such kits can be used for making labeled nucleic acidmolecules (single- or double-stranded). The kits of the inventioncomprise a carrier, such as a box or carton, having in close confinementtherein one or more containers, such as vials, tubes, bottles and thelike. In the kits of the invention, a first container contains one ormore of the reverse transcriptase enzymes of the invention (preferablyone or more such multi-subunit enzymes such as heterodimer enzymes ortwo subunit enzymes or variants, derivatives or mutants thereof) or oneor more of the compositions of the invention. The kits of the inventionmay also comprise, in the same or different containers, at least onecomponent selected from one or more DNA polymerases (preferablythermostable DNA polymerases), a suitable buffer for nucleic acidsynthesis and one or more nucleotides. Alternatively, the components ofthe kit may be divided into separate containers. In one aspect, the kitsof the invention comprise reverse transcriptases which have RNase Hactivity or are reduced or substantially reduced in RNase H activity.Such RTs preferably are selected from the group consisting of RSVreverse transcriptase, AMV reverse transcriptase, RAV reversetranscriptase, MAV reverse transcriptase and HIV reverse transcriptase.In additional preferred kits of the invention, the enzymes (reversetranscriptases and/or DNA polymerases) in the containers are present atworking concentrations.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following description of theinvention, and of the claims.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms used in recombinantDNA technology are utilized extensively. In order to provide a clear andmore consistent understanding of the specification and claims, includingthe scope to be given such terms, the following definitions areprovided.

Primer. As used herein, “primer” refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a nucleic acidmolecule.

Template. The term “template” as used herein refers to double-strandedor single-stranded nucleic acid molecules which are to be amplified,synthesized or sequenced. In the case of a double-stranded molecules,denaturation of its strands to form a first and a second strand ispreferably performed before these molecules may be amplified,synthesized or sequenced, or the double-stranded molecule may be useddirectly as a template. For single stranded templates, at least oneprimer, complementary to a portion of the template is hybridized underappropriate conditions and one or more polymerases or reversetranscriptases may then synthesize a nucleic acid molecule complementaryto all or a portion of said template. The newly synthesized molecules,according to the invention, may be equal or shorter in length than theoriginal template.

Incorporating. The term “incorporating” as used herein means becoming apart of a DNA and/or RNA molecule or primer.

Nucleotide. As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, andnucleotide derivatives that confer nuclease resistance on the nucleicacid molecule containing them. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (ddNTPs) and theirderivatives. Illustrated examples of dideoxyribonucleoside triphosphatesinclude, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.According to the present invention, a “nucleotide” may be unlabeled ordetectably labeled by well known techniques. Detectable labels include,for example, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels. Fluorescent labels ofnucleotides may include but are not limited fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham ArlingtonHeights, Ill.; Fluorescein-15-dATP. Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim Indianapolis, Ind.; and ChromaTide Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.

Oligonucleotide. “Oligonucleotide” refers to a synthetic or naturalmolecule comprising a covalently linked sequence of nucleotides whichare joined by a phosphodiester bond between the 3′ position of thedeoxyribose or ribose of one nucleotide and the 5′ position of thedeoxyribose or ribose of the adjacent nucleotide.

Hybridization. The terms “hybridization” and “hybridizing” refers tobase pairing of two complementary single-stranded nucleic acid molecules(RNA and/or DNA) to give a double-stranded molecule. As used herein, twonucleic acid molecules may be hybridized, although the base pairing isnot completely complementary. Accordingly, mismatched bases do notprevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used.

Probes. The term probes refer to single or double stranded nucleic acidmolecules or oligonucleotides which are detectably labeled by one ormore detectable markers or labels. Such labels or markers may be thesame or different and may include radioactive labels, fluorescentlabels, chemiluminescent labels, bioluminescent labels and enzymelabels, although one or more fluorescent labels (which are the same ordifferent) are preferred in accordance with the invention. Probes havespecific utility in the detection of nucleic acid molecules byhybridization and thus may be used in diagnostic assays.

Overview

The present invention provides kits, compositions and methods useful inovercoming the labeling limitations often observed during reversetranscription of nucleic acid molecules. Thus, the invention facilitatesthe production of labeled nucleic acid molecules (particularly cDNAmolecules) not heretofore possible.

In general, the invention provides compositions for use in reversetranscription of a nucleic acid molecule to produce labeled nucleic acidmolecules. Such compositions may comprise one or more reversetranscriptases (preferably one or more multi-subunit RTs). The enzymesin these compositions are preferably present in working concentrationsand have RNase H activity or are reduced or substantially reduced inRNase H activity, although mixtures of enzymes, some having RNase Hactivity and some reduced or substantially reduced in RNase H activity,may be used in the compositions of the invention. Preferred reversetranscriptases include RSV reverse transcriptase, AMV reversetranscriptase, RAV reverse transcriptase, MAV reverse transcriptase andHIV reverse transcriptase or other ASLV reverse transcriptases.

The invention is also directed to methods for reverse transcription ofone or more nucleic acid molecules comprising mixing one or more nucleicacid templates, which is preferably RNA or messenger RNA (mRNA) and morepreferably a population of mRNA molecules, with one or more polypeptideshaving reverse transcriptase activity (preferably multi-subunit RTs) andincubating the mixture under conditions sufficient to make one or morelabeled nucleic acid molecules complementary to all or a portion of theone or more templates. To make the nucleic acid molecule or moleculescomplementary to the one or more templates, at least one primer (e.g.,an oligo (dT) primer) and one or more nucleotides (a portion of whichare preferably labeled, most preferably fluorescently labeled) are usedfor nucleic acid synthesis in the 3′ to 5′ direction. Nucleic acidtemplates suitable for reverse transcription according to this aspect ofthe invention include any nucleic acid molecule, particularly thosederived from a prokaryotic or eukaryotic cell. Such cells may includenormal cells, diseased cells, transformed cells, established cells,progenitor cells, precursor cells, fetal cells, embryonic cells,bacterial cells, yeast cells, animal cells (including human cells),avian cells, plant cells and the like, or tissue isolated from a plantor an animal (e.g., human, cow, pig, mouse, sheep, horse, monkey,canine, feline, rat, rabbit, bird, fish, insect, etc.). Such nucleicacid molecules may also be isolated from viruses.

The invention also provides labeled nucleic acid molecules producedaccording to the above-described methods. Such labeled nucleic acidmolecules may be single or double stranded and are useful as detectionprobes. Depending on the labeled nucleotide(s) used during synthesis,the labeled molecules may contain one or a number of labels. Wheremultiple labels are used, the molecules may comprise a number of thesame or different labels. Thus, one type or multiple different labelednucleotides may be used during synthesis of nucleic acid molecules toprovide for the labeled nucleic acid molecules of the invention. Suchlabeled nucleic acid molecules will thus comprise one or more labelednucleotides (which may be the same or different).

The invention also provides kits for use in accordance with theinvention. Such kits comprise a carrier means, such as a box or carton,having in close confinement therein one or more container means, such asvials, tubes, bottles and the like, wherein the kit comprises, in thesame or different containers, one or more reverse transcriptases. Thekits of the invention may also comprise, in the same or differentcontainers, one or more DNA polymerases, one or more primers, one ormore suitable buffers and/or one or more nucleotides (such asdeoxynucleoside triphosphates (dNTPs) and preferably fluorescentlylabeled dNTP's).

In a preferred aspect, the RTs used in the invention comprise two ormore subunits (or derivatives, variants, fragments or mutants thereof)and preferably comprise two subunits (e.g., a dimer or heterodimer). Twosubunit reverse transcriptases typically have an α and a β subunitforming a dimer, although any form or combination of subunits (andderivatives, variants or mutants of such subunits) may be used. Suchcombinations may include αβ, ββ, αα and the like. Preferred two subunitRTs for use in the invention include HIV RT, RSV RT, AMV RT, AEV RT, RAVRT, HIV RT and MAV RT, or other ASLV RTs, or mutants, variants orderivatives thereof. In a preferred aspect, AMV RT and/or RSV RT is usedin accordance with the invention. Reverse transcriptases for use in theinvention may be obtained from natural or recombinant sources. See, forexample, published PCT application WO 98/47912. Alternatively, reversetranscriptases for use in the invention may be obtained commercially, orexample, from Life Technologies, Inc. (Rockville, Md.), Pharmacia(Piscataway, N.J.), Sigma (St. Louis, Mo.), or Boehringer MannheimBiochemicals (Indianapolis, Ind.). In a related aspect, at least onesubunit of the RTs of the invention may be modified or mutated to affectthe activity of the enzyme such as to reduce, substantially reduce oreliminate RNase H activity. Preferred RTs for use in the inventionincluding ThermoScript™ and ThermoScript™ II obtainable from LifeTechnologies, Inc. and others described in WO 98/47912 which isincorporated by reference in its entirety.

In accordance with the invention, the amount of labeled product ispreferably measured based on percent incorporation of the label ofinterest into synthesized product as may be determined by one skilled inthe art and as discussed in the Examples, although other means ofmeasuring the amount or efficiency of labeling of product will berecognized by one of ordinary skill in the art. The invention providesfor enhanced or increased percent incorporation of labeled nucleotideduring synthesis of a nucleic acid molecule from a template, preferablyduring synthesis of one or more cDNA molecules from RNA. According tothe invention, such enhancement or increase in percent incorporation ispreferably about equal to or greater than a 2-fold, a 5-fold, a 10-fold,a 15-fold, a 20-fold, a 25-fold, a 30-fold, a 40-fold or a 50-foldincrease or enhancement in percent incorporation compared to a standardreverse transcriptase such as MMLV RT and preferably SuperScript™ orSuperScript™ II available from Life Technologies, Inc. In anotheraspect, the percent incorporation of the labeled nucleotide (preferablya fluorescent nucleotide) during synthesis is equal to or greater thanabout 5%, equal to or greater than about 7.5%, equal to or greater thanabout 10%, equal to or greater than about 15%, equal to or greater thanabout 20%, equal to or greater than about 25% equal to or greater thanabout 30%, equal to or great than about 40% or equal to or greater thanabout 50%.

Enzymes for use in the invention may include those that are reduced orsubstantially reduced in RNase H activity. Such enzymes that are reducedor substantially reduced in RNase H activity may be obtained by mutatingthe RNase H domain within the reverse transcriptase of interest,preferably by one or more point mutations, one or more deletionmutations, and/or one or more insertion mutations as described above.See generally U.S. Pat. No. 5,668,005 and published PCT application WO9847912. By an enzyme “substantially reduced in RNase H activity” ismeant that the enzyme has less than about 30%, less than about 25%, lessthan about 20%, more preferably less than about 15%, less than about10%, less than about 7.5%, or less than about 5%, and most preferablyless than about 5% or less than about 2%, of the RNase H activity of thecorresponding wildtype or RNase H⁺ enzyme such as wildtype MoloneyMurine Leukemia Virus (M-MLV), Avian Myeloblastosis Virus (AMV) or RousSarcoma Virus (RSV) reverse transcriptases. The RNase H activity of anyenzyme may be determined by a variety of assays, such as thosedescribed, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, M. L.,et al., Nucl. Acids Res. 16:265 (1988), in Gerard, G. F., et al., FOCUS14(5):91 (1992), and in U.S. Pat. No. 5,668,005, the disclosures of allof which are fully incorporated herein by reference.

Preferred enzymes for use in the invention include, but are not limitedto, RSV H⁻ reverse transcriptase, AMV H⁻ reverse transcriptase, RAV H⁻reverse transcriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase (see generally WO 98/47912). Particularly preferredenzymes used in the invention include ThermoScript™ and ThermoScript™ IIobtainable from Life Technologies, Inc. It will be understood by one ofordinary skill, however, that any enzyme capable of producing a DNAmolecule from a ribonucleic acid molecule (i.e., having reversetranscriptase activity) that is substantially reduced in RNase Hactivity may be equivalently used in the compositions, methods and kitsof the invention.

A variety of DNA polymerases are useful in accordance with the presentinvention. Such polymerases include, but are not limited to, Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neapolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosis (Pfu) DNA polymerase, DEEPVENT™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, Mycobacterium spp. DNA polymerase (Mtb, Mlep), andmutants, variants and derivatives thereof.

DNA polymerases used in accordance with the invention may be any enzymethat can synthesize a DNA molecule from a nucleic acid template,typically in the 5′ to 3′ direction. Such polymerases may be mesophilicor thermophilic, but are preferably thermophilic. Mesophilic polymerasesinclude T5 DNA polymerase, T7 DNA polymerase (Wiemann, S., et al.,BioTechnique 18:688 (1995) and Voss, H., et al., BioTechnique 23:312(1997)), Klenow fragment DNA polymerase, DNA polymerase III, and thelike. Preferred DNA polymerases are thermostable DNA polymerases such asTaq (Voss, H., et al., BioTechnique 23:312 (1997)), Tne, Tma, Pfu,VENT™, DEEPVENT™, Tth (Chang, H., et al., J. Immuno. Methods 176:235(1994)) and mutants, variants and derivatives thereof (U.S. Pat. No.5,436,149; U.S. Pat. No. 5,512,462; WO 92/06188; WO 92/06200; WO96/10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al.,PCR Meth. Appl. 2:275-287 (1993); Flaman, J.-M., et al., Nucl. AcidsRes. 22(15):3259-3260 (1994)). For amplification of long nucleic acidmolecules (e.g., nucleic acid molecules longer than about 3-5 Kb inlength), at least two DNA polymerases (one substantially lacking 3′exonuclease activity and the other having 3′ exonuclease activity) aretypically used. See U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462;and Barnes, W. M., Gene 112:29-35 (1992), the disclosures of all ofwhich are incorporated herein in their entireties.

Formulation of Enzyme Compositions

To form the compositions of the present invention, one or more reversetranscriptases are preferably admixed in a buffered salt solution. Oneor more DNA polymerases and/or one or more nucleotides (preferablyincluding one or more fluorescent nucleotides which may be the same ordifferent) may optionally be added to make the compositions of theinvention. The compositions of the invention may also comprise one ormore nucleic acid templates and/or one or more primers. More preferably,the enzymes are provided at working concentrations in stable bufferedsalt solutions. The terms “stable” and “stability” as used hereingenerally mean the retention by a composition, such as an enzymecomposition, of at least 70%, preferably at least 80%, and mostpreferably at least 90%, of the original enzymatic activity (in units)after the enzyme or composition containing the enzyme has been storedfor about one week at a temperature of about 4° C., about two to sixmonths at a temperature of about −20° C., and about six months or longerat a temperature of about −80° C. As used herein, the term “workingconcentration” means the concentration of an enzyme that is at or nearthe optimal concentration used in a solution to perform a particularfunction (such as reverse transcription of nucleic acids).

The water used in forming the compositions of the present invention ispreferably distilled, deionized and sterile filtered (through a 0.1-0.2micrometer filter), and is free of contamination by DNase and RNaseenzymes. Such water is available commercially, for example from SigmaChemical Company (Saint Louis, Mo.), or may be made as needed accordingto methods well known to those skilled in the art.

In addition to the enzyme components, the present compositionspreferably comprise one or more buffers and cofactors necessary forsynthesis of a labeled nucleic acid molecule such as a cDNA molecule.Particularly preferred buffers for use in forming the presentcompositions are the acetate, sulfate, hydrochloride, phosphate or freeacid forms of Tris-(hydroxymethyl)aminomethane (TRIS®), althoughalternative buffers of the same approximate ionic strength and pKa asTRIS® may be used with equivalent results. In addition to the buffersalts, cofactor salts such as those of potassium (preferably potassiumchloride or potassium acetate) and magnesium (preferably magnesiumchloride or magnesium acetate) are included in the compositions.Addition of one or more carbohydrates and/or sugars to the compositionsand/or synthesis reaction mixtures may also be advantageous, to supportenhanced stability of the compositions and/or reaction mixtures uponstorage. Preferred such carbohydrates or sugars for inclusion in thecompositions and/or synthesis reaction mixtures of the inventioninclude, but are not limited to, sucrose, trehalose, and the like.Furthermore, such carbohydrates and/or sugars may be added to thestorage buffers for the enzymes used in the production of the enzymecompositions and kits of the invention. Such carbohydrates and/or sugarsare commercially available from a number of sources, including Sigma(St. Louis, Mo.).

It is often preferable to first dissolve the buffer salts, cofactorsalts and carbohydrates or sugars at working concentrations in water andto adjust the pH of the solution prior to addition of the enzymes. Inthis way, pH-sensitive enzymes will be less subject to acid- oralkaline-mediated inactivation during formulation of the presentcompositions.

Concentrations of the RTs in the compositions of the invention may varydepending on the type of reverse transcriptase used. For example, AMVRTs, MAV RTs, RSV RTs and RAV RTs are preferably added at a workingconcentration in the solution of about 100 to about 5000 units permilliliter, about 125 to about 4000 units per milliliter, about 150 toabout 3000 units per milliliter, about 200 to about 2500 units permilliliter, about 225 to about 2000 units per milliliter, and mostpreferably at a working concentration of about 250 to about 1000 unitsper milliliter. The enzymes in the thermophilic DNA polymerase group andmutants, variants and derivatives thereof are preferably added at aworking concentration in the solution of about 100 to about 1000 unitsper milliliter, about 125 to about 750 units per milliliter, about 150to about 700 units per milliliter, about 200 to about 650 units permilliliter, about 225 to about 550 units per milliliter, and mostpreferably at a working concentration of about 250 to about 500 unitsper milliliter. The enzymes may be added to the solution in any order,or may be added simultaneously.

The compositions of the invention may further comprise one or morenucleotides (preferably a portion of which are fluorescent nucleotides),which are preferably deoxynucleoside triphosphates (dNTPs). The dNTPcomponents of the present compositions serve as the “building blocks”for newly synthesized nucleic acids, being incorporated therein by theaction of the polymerases or reverse transcriptases.

Production of Nucleic Acid or cDNA Molecules

In accordance with the invention, nucleic acid or cDNA molecules(single-stranded or double-stranded) may be prepared from a variety ofnucleic acid template molecules. Preferred templates for use in thepresent invention include single-stranded or double-stranded DNA and RNAmolecules, as well as double-stranded DNA:RNA hybrids. More preferredtemplates include messenger RNA (mRNA), transfer RNA (tRNA) andribosomal RNA (rRNA) molecules, although mRNA molecules are thepreferred template according to the invention.

Preferably the nucleic acid templates may be obtained from naturalsources, such as a variety of cells, tissues, organs or organisms. Cellsthat may be used as sources of nucleic acid molecules may be prokaryotic(bacterial cells, including but not limited to those of species of thegenera Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic(including fungi (especially yeasts), plants, protozoans and otherparasites, and animals including insects (particularly Drosophila spp.cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human cells)).

Mammalian somatic cells that may be used as sources of nucleic acidsinclude blood cells (reticulocytes and leukocytes), endothelial cells,epithelial cells, neuronal cells (from the central or peripheral nervoussystems), muscle cells (including myocytes and myoblasts from skeletal,smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells. Also suitable for use as nucleic acidsources are mammalian tissues or organs such as those derived frombrain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous,skin, genitourinary, circulatory, lymphoid, gastrointestinal andconnective tissue sources, as well as those derived from a mammalian(including human) embryo or fetus.

Any of the above prokaryotic or eukaryotic cells, tissues and organs maybe normal, diseased, transformed, established, progenitors, precursors,fetal or embryonic. Diseased cells may, for example, include thoseinvolved in infectious diseases (caused by bacteria, fungi or yeast,viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, muscular dystrophy ormultiple sclerosis) or in cancerous processes. Transformed orestablished animal cell lines may include, for example, COS cells, CHOcells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293cells, L929 cells, F9 cells, and the like. Other cells, cell lines,tissues, organs and organisms suitable as sources of nucleic acids foruse in the present invention will be apparent to one of ordinary skillin the art.

Once the starting cells, tissues, organs or other samples are obtained,nucleic acid templates (such as mRNA) may be isolated therefrom bymethods that are well-known in the art (See, e.g., Maniatis, T., et al.,Cell 15:687-701 (1978); Okayama, H., and Berg, P., Mol. Cell. Biol.2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene 25:263-269(1983)). The nucleic acid molecules thus isolated may then be used toprepare cDNA molecules and cDNA libraries in accordance with the presentinvention.

In the practice of the invention, labeled cDNA molecules or labeled cDNAlibraries are produced by mixing one or more nucleic acid moleculesobtained as described above, which is preferably one or more mRNAmolecules such as a population of mRNA molecules, with one or morepolypeptides having reverse transcriptase activity of the invention, orwith one or more of the compositions of the invention and preferablywith one or more of the RSV RTs and/or AMV RTs and/or other ASLV RTs ofthe invention, under conditions favoring the reverse transcription ofthe nucleic acid molecule by the action of the enzymes or thecompositions to form one or more labeled cDNA molecule (single-strandedor double-stranded). Thus, the method of the invention comprises (a)mixing one or more nucleic acid templates (preferably one or more RNA ormRNA templates, such as a population of mRNA molecules) with one or morereverse transcriptases of the invention and (b) incubating the mixtureunder conditions sufficient to make one or more labeled nucleic acidmolecules complementary to all or a portion of the one or moretemplates. The invention may be used in conjunction with methods of cDNAsynthesis such as those described in the Examples below, or others thatare well-known in the art (see, e.g., Gubler, U., and Hoffman, B. J.,Gene 25:263-269 (1983); Krug, M. S., and Berger, S. L., Meth. Enzymol.152:316-325 (1987); Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 8.60-8.63 (1989)), to produce cDNA molecules orlibraries.

In other aspects, the invention may be used in methods for amplifyingnucleic acid molecules. Nucleic acid amplification methods according tothis aspect of the invention may be one-step (e.g., one-step RT-PCR) ortwo-step (e.g., two-step RT-PCR) reactions. According to the invention,one-step RT-PCR type reactions may be accomplished in one tube therebylowering the possibility of contamination. Such one-step reactionscomprise (a) mixing a nucleic acid template (e.g., mRNA) with one ormore polypeptides having reverse transcriptase activity of the inventionand with one or more DNA polymerases and (b) incubating the mixtureunder conditions sufficient to amplify a labeled nucleic acid moleculecomplementary to all or a portion of the template. Alternatively,amplification may be accomplished by mixing a template with one or morepolypeptides having reverse transcriptase activity of the invention.Incubating such a reaction mixture under appropriate conditions allowsamplification of a labeled nucleic acid molecule complementary to all ora portion of the template. Such amplification may be accomplished by thereverse transcriptase activity alone or in combination with a DNApolymerase. Two-step RT-PCR reactions may be accomplished in twoseparate steps. Such a method comprises (a) mixing a nucleic acidtemplate (e.g., mRNA) with one or more reverse transcriptases of theinvention, (b) incubating the mixture under conditions sufficient tomake a labeled nucleic acid molecule (e.g., a DNA molecule)complementary to all or a portion of the template, (c) mixing thelabeled nucleic acid molecule with one or more DNA polymerases and (d)incubating the mixture of step (c) under conditions sufficient toamplify the labeled nucleic acid molecule. For amplification of longnucleic acid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, such as one DNA polymerasehaving 3′ exonuclease activity and another DNA polymerase beingsubstantially reduced in 3′ exonuclease activity.

Amplification methods which may be used in accordance with the presentinvention include PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202), StrandDisplacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0 684 315),and Nucleic Acid Sequence-Based Amplification (NASBA; U.S. Pat. No.5,409,818; EP 0 329 822).

Kits

In another embodiment, the present invention may be assembled into kitsfor use in reverse transcription or amplification of a nucleic acidmolecule. Kits according to this aspect of the invention comprise acarrier means, such as a box, carton, tube or the like, having in closeconfinement therein one or more container means, such as vials, tubes,ampules, bottles and the like, wherein a first container means containsone or more polypeptides of the invention having reverse transcriptaseactivity. The kits of the invention may also comprise (in the same orseparate containers) one or more DNA polymerases, a suitable buffer, oneor more nucleotides (preferably including one or more fluorescentnucleotides which may be the same or different) and/or one or moreprimers.

In a specific aspect of the invention, the reverse transcription andamplification kits may comprise one or more components (in mixtures orseparately) including one or more polypeptides having reversetranscriptase activity of the invention, one or more nucleotides neededfor synthesis of a labeled nucleic acid molecule, and/or one or moreprimers (e.g., oligo(dT) for reverse transcription). Such reversetranscription and amplification kits may further comprise one or moreDNA polymerases. Preferred polypeptides having reverse transcriptaseactivity, DNA polymerases, nucleotides, primers and other componentssuitable for use in the reverse transcription and amplification kits ofthe invention include those described herein. The kits encompassed bythis aspect of the present invention may further comprise additionalreagents and compounds necessary for carrying out standard nucleic acidreverse transcription or amplification protocols. Such kits may alsocomprise instructions for labeling nucleic acid molecules in accordancewith the invention.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications aid adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES First Strand cDNA Synthesis Using Fluorescent Nucleotide

For the SuperScript™ II reactions, Cy3-dUTP or R110-dUTP wasincorporated into cDNA using 1 μg of Hela mRNA, primed with 1 μg ofoligo d(T) 25mer. But for the AMV RT and ThermoScript™ reactions, 0.5 μgof oligo d(T) 25 mer was used to prime 1 μg Hela mRNA. This mixture washeated to 70° C. for 10 min, and then transferred to ice for 10 min. Thereaction conditions varied between the reverse transcriptases. TheSuperScript II™ reaction buffer contained 50 mM Tris-HCl (pH 8.3), 75 mMKCl, 3 mM MgCl₂, 10 mM DTT, 1 μCi P³² α-dCTP, 500 μM dNTPs, and 100 μMfluorescent dUTP. The ThermoScript™ reaction buffer contained 50 mMTris-HCl (pH 8.4), 75 mM KCl, 7.5 mM MgCl₂, 10 mM DTT, 2 μCi P³² α-dCTP,1 mM dNTPs, and 200 μM fluorescent dUTP. The AMV RT reaction buffercontained 100 mM Tris-HCl (pH 8.3), 100 mM KCl, 10 mM MgCl₂, 10 mM DTT,0.5 μCi P³² α-dCTP, 250 μM dNTPs, and 100 μM fluorescent dUTP. Thereaction buffer was added to the Hela mRNA and oligo d(T) 25mer mixture.Two hundred units of SuperScript II™, 15 units of ThermoScript™, or 7.5units of AMV RT was used in the reaction. Then the reaction wasincubated at the RT's optimal reaction temperature (45° C. forSuperScript™ II, 55° C. for ThermoScript™ and 42° C. for AMV RT) for onehour. Two microliters of the reaction mixture was pippeted and mixedwith 5 μg of yeast tRNA in a final of 50 μl of 20 mM EDTA solution. ThisEDTA solution was used in the first strand cDNA yield calculation.

TCA Precipitation and First Strand cDNA Synthesis Yield Calculation

Duplicate 10 μl samples (from 20 mM EDTA solution) were spotted ontoglass fiber filters and dried under a heat lamp for 15 min. One set ofthe filters was washed once with ice-cold 10% (w/v) TCA solution andtwice with 5% TCA solution (10 min. perwash) at room temperature. Afterthe washes, the filters were washed with 95% ethanol for 5 min. and thendried under a heat lamp. The washed and unwashed filters were counted instandard scintillant to determine the amount of P³² in the reaction, aswell as the amount of P³² that was incorporated. The equation used forthe calculation of first strand synthesis yield is as follows:

Specific Activity (SA; cpm/pmol dCTP)=cpm of 10 μl from unwashed sample200 pmol dCTP

Amount of cDNA (μg)=cpm of washed sample×50×(4 pmole dNTP/pmole dCTP)SA×(3,030 pmole dNTP/μg cDNA

Absorbance for Fluorescent Nucleotide Incorporation

Two to four microliters of the first strand reaction mixture was saved,this was used as a “standard”, and the remaining 16-18 μl of the firststrand reaction mixture was precipitated with one half volume of NH₄Acand 2.5-fold volume of ethanol, and 5 μg of yeast tRNA as a carrier.Following precipitation, the pellet was washed twice with 70% ethanol tolower background fluorescence. The precipitates were resuspended in 50mM Tris buffer (pH 7.5), and the fluorescent nucleotide incorporationwas determined. The “standard” was diluted in 50 mM Tris buffer and theabsorbance determined. The excitation spectra for Cy3-dUTP and R110-dUTPare 550 nm and 503 nm. The emission spectra ranges are 560-600 nm forCy3-dUTP and 508-560 nm for R110-dUTP. The difference between the“standard” and the ethanol precipitated sample is the fluorescentnucleotide incorporation.

Calculation of Percent Incorporation Sample from SS II, TS I withCy3-dUTP/HeLa mRNAA. Standard Curve, which is a Series Dilution of Standard SampleContaining 200 μM of Cy3-dUTP.

Light units, peak of Convert Dilution emission concentration, nM 1:1,0001,796,330 200 1;2,000 822,791 100 1:4,000 437,004 50 1:8,000 221,297 251:16,000 114,387 12.5 1:32,000 59,297 6.25 1:64,000 30,642 3.13B. Sample of SS II with Cy3-dUTP/Hela mRNA

The unprecipitated reaction sample contained 100 p mole/μl of Cy3-dUTP,which calculated from the concentration of 100 μM Cy3-dUTP in 1 μl.

The precipitated reaction sample (total of 14 μl), which containedCy3-dUTP incorporated cDNA, will be measured in emission (14 μl). Thelight units will be converted to nM by the standard curve.

14  µl  of  SS  II  w/Cy3-dUTP  (100  µM  in  reaction) = 97,347  light  units = 13  nM  (in  2  ml  vol)The  p  mole  conversion:  13  nM × (2/1000  liter) = 26  p  mole  (in  14  µl) = 1.86  p  mole/µ l

1.86/100=1.86% (incorporation)C. Sample of TS I with Cy3-dUTP/Hela mRNA

The unprecipated reaction sample contained 200 p mole/μl of Cy3-dUTP.

The precipitated reaction sample (2 μl) will be measured in emission.

2  µl  of  TS  I  w/Cy3-dUTP  (200  µM  in  reaction) = 282,729  light  units = 34  nM  (in  2  ml  vol)The  p  mole  conversion:  34  nM × (2/1000  liter) = 68  p  mole  (in  2  µl) = 34  p  mole/µl

34/200=17% (incorporation)Comparison of SuperScript™ II (SS II) and ThermoScript™ (TS I) inCy3-dUTP incorporation in 2.3 Kb control RNA and MAP4 RNA (5.0 Kb).

cDNA Fluorescent synthesis Cy3-dUTP nucleotide incorp. Yield (%) Incorp.(%) Increased (fold) SS II & 2.3 Kb RNA 20.1 1.3 — TS I & 2.3 Kb RNA23.0 9.4  7.2 SS II & MAP4 RNA 33.0 1.2 — TS I & MAP4 RNA 38.6 18.4 15.3Note: The cDNA synthesis yield with normal dNTP are 28.8% for SS II,33.3% for TS I.Comparison of SS II, TS I, ThermoScript™ II (TS 2) and AMV RT inCy3-dUTP and Rh110-dUTP incorporation in HeLa m-RNA.

cDNA Fluorescent Synthesis Cy3-dUTP nucleotide incorp. Yield (%) Incorp.(%) Increased (fold SS II & HeLa/Cy3dU 27.9 1.86 — TS I & HeLa/Cy3dU21.0 17.0 9.1 TS II & HeLa/Cy3dU 21.0 19.4 10.4  AMV & HeLa/Cy3dU 20.320.5 11.0  SS II & HeLa/R110dU 19.8 2.57 — TS I & HeLa/R110dU 14.9 20.678.0 TS II & HeLa/R110dU 13.1 25.33 9.9 AMV & HeLa/R110dU 10.0 24.5 9.5Note: The cDNA synthesis yield with normal dNTP are 30.7% for SS II,26.1% for TS I, 26.9% for TS II, and 22.1% for AMV RT.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. A composition for use in labeling one or more nucleic acid molecules,said composition comprising one or more enzymes having reversetranscriptase activity, wherein said enzymes are multi-subunit enzymes.2. The composition of claim 1, wherein said enzymes are heterodimers. 3.The composition of claim 1, wherein said enzymes are selected from thegroup consisting of ASLV reverse transcriptases.
 4. The composition ofclaim 1, wherein said enzymes are reduced or substantially reduced inRNase H activity.
 5. The composition of claim 1, wherein said enzymesare selected from the group consisting of RSV reverse transcriptase, AMVreverse transcriptase, RAV reverse transcriptase, MAV reversetranscriptase and HIV reverse transcriptase, and derivatives, variants,fragments or mutants thereof.
 6. The composition of claim 1, whereinsaid composition further comprises one or more fluorescently labelednucleotides.
 7. The composition of claim 6, wherein said nucleotides arelabeled with a fluorescent molecule or marker selected from the groupconsisting of rhodamine, fluoroscein, Cy3, and Cy5.
 8. The compositionof claim 1, wherein said composition further comprises one or more DNApolymerases, and mutants, fragments, variants and derivatives thereof.9. The composition of claim 1, wherein said composition furthercomprises a reverse transcription buffer and/or a nucleic acid template.10. A method for reverse transcription of one or more nucleic acidmolecules comprising (a) mixing one or more nucleic acid templates withone or more enzymes having reverse transcriptase activity, wherein saidenzymes are multi-subunit enzymes; and (b) incubating said mixture underconditions sufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of said one or more templates, whereinat least one of said first nucleic acid molecules is detectably labeled.11. The method of claim 10, wherein said nucleic acid template is amessenger RNA (mRNA) molecule or a population of mRNA molecules.
 12. Themethod of claim 10, said method further comprising incubating said oneor more first nucleic acid molecules under conditions sufficient to makeone or more second nucleic acid molecules complementary to all or aportion of said one or more first nucleic acid molecules.
 13. The methodof claim 10, wherein said enzymes are selected from the group consistingof ASLV reverse transcriptases.
 14. The method of claim 10, wherein saidenzymes are selected from the group consisting of RSV RT, AMV RT,ThermoScript™ and ThermoScript™ II.
 15. The method of claim 10, whereinsaid detectable label is a fluorescent label.
 16. The method of claim10, wherein said fluorescent label is selected from the group consistingof fluorescein, rhodamine, Cy3 and Cy5.
 17. The method of claim 10,wherein said detectably labeled first nucleic acid molecules compriseone or more fluorescently labeled nucleotides.
 18. The method of claim10, wherein said detectable labels are the same or different.
 19. Themethod of claim 18, wherein said detectable labels are fluorescentlabels.
 20. A labeled nucleic acid molecules prepared according to themethod of claim
 10. 21. A kit for use in labeling one or more nucleicacid molecules, said kit comprising one or more enzymes having reversetranscriptase activity, wherein said enzymes are multi-subunit enzymes.22. The kit of claim 21, said kit further comprising one or morecomponents selected from the group consisting of one or morenucleotides, one or more DNA polymerases, a suitable buffer, and one ormore primers.
 23. The kit of claim 22, wherein at least one or saidnucleotides are fluorescent nucleotides.